The increasing demand for monoclonal antibodies (MABs) useful in research, diagnosis, therapy, and purification purposes has created a need to optimize protein production techniques. The prior art includes improved bioreactor designs and bioreactor operation to increase cell densities or the longevity of the cell culture by nutrient feedings.
Bioreactors have been operated in fed-batch, immobilized, perfusion and continuous modes. Alternate strategies, such as the use of temperature, media formulation, including the addition of mouse peritoneal factors, growth inhibitors, autocrine factors or cyclic mononucleotides and hyperstimulation by osmolarity stress, have been used to enhance protein production. These approaches have shown only marginal success.
Commonly used basal cell culture media are RPMI 1640, DMEM (Dulbecco's modified Eagle's medium), Ham's F12 and DMEM/F12 (DF). A modified medium, eRDF, prepared from RDF (RPMI:DMEM:F12, 2:1:1) by enrichment with amino acids, glucose and vitamins, was described by Murakami, H. (1989) “Serum-free media used for cultivation of hybridomas.” In: A. Mizrahi (Ed.), Advances in Biotechnological Processes, Vol. 11, Monoclonal Antibodies: Production and Application. Alan R. Liss, New York, pp. 107-141. Murakami showed that doubling the total amino acids or glucose alone in the culture medium did not increase cell density, but concurrent elevation of amino acids and glucose maximized the cellular growth by threefold.
Hyper-stimulation of monoclonal antibody production by high osmolarity stress in an eRDF medium is described in Chua, F., et al. (1994) J. Immunological Methods 167:109-119, and Chua, F., et al. (1994) J. Biotechnology 37:265-275. However, the maximum IgG concentration achieved was about 300 μg/mL and 270 μg/mL for HG11 and TBC3 cells, respectively, at medium osmolarities about 350 to 400 mOsm. Further increase in osmolarity of the therein-described media with NaCl caused deterioration in antibody production.
Oh, S. K. W., et al. ((1995) Biotechnology and Bioengineering 48:525-535) report that hybridomas increased metabolic activities and amino acids uptake via the Na+ dependent symports to compensate for the osmotically elevated external environment.
Oh, S. K. W., et al. ((1996) “Flow Cytometric Studies of Osmotically Stressed and Sodium Butyrate-Treated Hybridoma Cells” in Flow Cytometry Applications in Cell Culture, Marcel Dekker, Inc., (Eds. M. Al-Rubeai and A. N. Emergy) New York, Base1, Hong Kong, pp. 101-119) also describes the application of flow cytometry in examining the relationships between total cellular monoclonal antibody content, cell size, and cell cycle distribution of hybridomas subjected to environmental stress.